To investigate whether vitamin B6 supplementation has a beneficial effect on immune responses in critically ill patients.
A single-blind intervention study.
The study was performed at the Taichung Veterans General Hospital, the central part of Taiwan.
Fifty-one subjects who stayed over 14 days in the intensive care unit completed the study. Subjects were not treated with any vitamin supplement before the intervention.
Patients were randomly assigned to one of three groups, control (n=20), a daily injection of 50 mg vitamin B-6 (B6-50, n=15), or 100 mg vitamin B-6 (B6-100, n=16) for 14 days.
Main outcome measures:
Plasma pyridoxal 5′-phosphate (PLP), pyridoxal (PL), 4-pyridoxic acid (4-PA), erythrocyte alanine (EALT-AC) and aspartate (EAST-AC) aminotransaminase activity coefficient, and urinary 4-PA were measured. The levels of serum albumin, hemoglobin, hematocrit, high-sensitivity C-reactive protein (hs-CRP) and immune responses (white blood cell, neutrophils, total lymphocytes count (TLC), T- (CD3) and B-(CD19) lymphocytes, T-helper (CD4) and suppressor (CD8) cells) were determined.
Plasma PLP, PL, 4-PA and urinary 4-PA concentrations significantly increased in two treated groups. T-lymphocyte and T-helper cell numbers and the percentage of T-suppressor cell significantly increased on day 14 in the B6-50 group. Total lymphocyte count, T-helper and T-suppressor cell numbers, the percentage of T-lymphocyte cells and T-suppressors significantly increased in the B6-100 group at the 14th day. There were no significant changes with respect to immune responses in the control group over 14 days.
A large dose of vitamin B6 supplementation (50 or 100 mg/day) could compensate for the lack of responsiveness of plasma PLP to vitamin B6 intake, and further increase immune response of critically ill patients.
This study was supported by the National Science Council, Taiwan, Republic of China (NSC-92-2320-B-040-026).
Although the mechanism of plasma pyridoxal 5′phosphate (PLP), the physiological active coenzyme form of vitamin B6, on immune responses has not yet been ascertained, the deficiency of PLP has been demonstrated to significantly impair both humoral and cell-mediated immunity (Kumar and Axelrod, 1968; Axlerod, 1971; Sergeev et al., 1978; Willis-Carr and St Pierre, 1978; Ha et al., 1984). Casciato et al. (1984) indicated that eight hemodialysis patients supplemented with 50 mg/day of vitamin B6 (pyridoxine hydrochloride (HCl)) for 3–5 weeks showed an improvement in their nitroblue tetrazolium reduction test, the generation of chemotactic factors from plasma, lymphocyte subpopulation and lymphocyte transformation in response to mitogens. Lymphocyte proliferation significantly increased in response to phytohemagglutinin, pokweweed and Staphylococcus aureus after 11 healthy elderly were supplemented with 50 mg/day of vitamin B6 (pyridoxine) for 2 months; Talbott et al. (1987), therefore, indicated that increasing vitamin B6 intake could improve immune responsiveness of both T and B cells. Folkers et al. (1993) treated nine healthy subjects with 300 mg/day of pyridoxine, consequently their T4 lymphocytes and T4/T8 ratio significantly increased over 2 months. The improvements of immune response seemed to occur after high doses of vitamin B6 supplements (>50 mg/day pyridoxine) even though subjects had no any evidence of vitamin B6 deficiency.
Our previous study (Huang et al., 2002) indicated that although critically ill patients had an adequate vitamin B6 intake (1.6 mg/day for men and women who are older than 51 years, Taiwan Dietary Reference Intakes, Department of Health, Taiwan, 2002), their plasma PLP and pyridoxal (PL) still significantly decreased during their stay in the intensive care unit (ICU). Critically ill patients were under severe stress, inflammation, and clinical conditions, which may increase the utilization and metabolic turnover of plasma PLP or even cause the redistribution of PLP from plasma to erythrocyte (Louw et al., 1992; Talwar et al., 2003a; Huang et al., 2005). It thus would be interesting to know whether a high dose of vitamin B6 supplement would increase the immune response of critically ill patients. The purpose of this study was to investigate whether vitamin B6 supplementation has a beneficial effect on immune responses in critically ill patients.
Materials and methods
A single-blind study was conducted at the Taichung Veteran General Hospital, which is a teaching hospital in the central part of Taiwan. One hundred and twelve patients were admitted or transferred to the ICU screened from December 2003 to December 2004. Patients were excluded if they were uremic or clinically unstable or unconscious at the time of entry to the study. Patients were also excluded if they were given additional multivitamin supplements based on a physician's decision. All patients received either enteral, total parenteral, or combined (enteral plus total parenteral) nutritional support based on the physician's recommendations. Daily macronutrients (carbohydrate, fat, and protein) and vitamin B6 intake from nutritional support and intravenous crystalloid infusions were recorded routinely by the ICU nurses and dietitians. The diagnoses, age, sex, and height were obtained from the medical records. Patient's weight, body mass index (BMI; kg/m2), and the severity of illness (by using APACHE II score) on admission were assessed within 24 h of admission and again on day 14 in the ICU. Only patients requiring at least 14 days of mechanical ventilation were included; therefore, 51 patients (41 men and 10 women) with the mean age of 70.2±14.5 years successfully completed the study after informed consent was obtained. The study was approved by the Committee for Ethics of Chung Shan Medical University.
Patients were randomly assigned to either the control (no vitamin B6 injection, n=20), 50 mg vitamin B6 (B6-50) (n=16), or 100 mg vitamin B6 group (B6-100) (n=15). Patients in the groups of B6-50 and B6-100 were receiving the injection of vitamin B6 daily by the ICU nurse for 14 days. The vitamin B6 injection (pyridoxine HCl) was commercially available (Ying Yuan Chemical Pharmaceutical Co., Ltd, Tainan, Taiwan).
Fasting venous blood samples were collected in Vacutainer tubes (Becton Dickinson, Rutherford, NJ, USA) containing an ethylenediaminetetraacetic acid as an anticoagulant or no anticoagulant as required to determine hematological (i.e, white blood cell (WBC), total lymphocyte count (TLC), neutrophils, hemoglobin, hematocrit, albumin, prealbumin, creatinine, alkaline phosphatase, high-sensitivity C-reactive protein (hs-CRP), vitamin B6 status (i.e., plasma PLP, PL, 4-pyridoxic acid (4-PA), erythrocyte alanine aminotransferase, and erythrocyte aspartate aminotransferase activity coefficient (EALT-AC and EAST-AC), and T-cell subsets (i.e., CD3, CD4, CD8, and CD19 antigens). During the intervention period, blood samples were taken on the 1st and 14th days in the ICU. Blood samples were transported on ice and separated into plasma (or serum) and red blood cells within 1 h by low-speed centrifugation (2500 r.p.m., 15 min). Samples were then stored frozen (−80°C) until analysis.
Plasma PLP, PL, and 4-PA were determined by high-performance liquid chromatography as previously described (Talwar et al., 2003b). The fluorescence detector's excitation and emission wavelengths were 320 and 420 nm, respectively. The intra-assay of plasma PLP, PL, and 4-PA variabilities were 2.6% (n=5), 3.8% (n=5), and 1.5% (n=5), respectively. The inter-assay of plasma PLP, PL, and 4-PA variabilities were 4.2% (n=8), 4.8% (n=8), and 2.1% (n=8), respectively. EALT and EAST with and without PLP stimulation in vitro were measured by the method of Woodring and Storvick (1970). All EALT and EAST activity measurements were performed by using fresh erythrocyte samples collected on the day of analysis. Plasma B6 concentrations and transaminase activity measurements were carried out under yellow light to prevent photodestruction. All analyses were performed in duplicate. Vitamin B6 deficiency was defined as plasma PLP concentration <20 nmol/l, EALT-AC >1.25 and/or EAST-AC >1.8 (Leklem, 1990; Food and Nutrition Board, 1998).
Lymphocyte subsets were analyzed by flow cytometry (FACS Calibur, Becton Dickinson, San Jose, CA, USA) using fluorescent-labeled antibodies specific to the cell markers. All tests were performed within 48 h of sampling. T-lymphocyte (CD3+) and B-lymphocyte (CD19+) percentages were determined with fluorescein isothiocyanate (FITC)-labeled CD3 (Leu 4), clone SK7 and phycoerythrin (PE)-labeled CD 19 (Leu-12), clone 4G7 (Becton Dickinson, Immunocytometry Systems, San Diego, CA, USA). T-helper (CD4+) and T-suppressor (CD8+) lymphocyte percentages were determined with FITC-labeled CD4 (Leu 3a), clone SK3 and PE-labeled CD8 (Leu-2a), clone SK1 (Becton Dickinson, Immunocytometry Systems, San Diego, CA, USA). The following values were considered as the reference ranges for our laboratory: WBC, 5000–10000/mm3; neutrophil, 3000–7000/mm3 or 60–70%; TLC, 1000–4000/mm3 or 20–40%; T lymphocyte, 61–76%; B lymphocyte (CD 19), 6–22%; T-helper cell, 27–43%; and T-suppressor cell (CD8), 24–35% (Huppert et al., 1998).
Data was analyzed using the SigmaStat statistical software (version 2.03; Jandel Scientific, San Rafael, CA, USA). The paired t-test or Wilcoxon signed rank test was used to compare significant differences in demographic and health characteristics and the data for biochemical measurements within each group between the 1st and the 14th day. Among groups, one-way analysis of variance (ANOVA) or Kruskal–Wallis one-way analysis on ranks was used to compare differences with values at day 1 and day 14. Spearmen correlation coefficients were used to assess the relationship between vitamin B6 status parameters and immune indices. Statistical results were considered to be significant at P<0.05. Values presented in the text are means±standard deviation (s.d.).
Characteristics of subjects
Demographic and health characteristics of subjects in each group are shown in Table 1. There were 37 surgical and 14 medical patients. Patients' age ranged from 24 to 91 years, with a mean age of 70.2 years. The three groups of patients were well matched for age, weight, and severity of illness (APACHE II score). However, patients in the control group were significantly taller; therefore, the BMI value was expected to be significantly lower than patients in the other two groups. The APACHE II score of patients in the B6-50 group significantly decreased on the 14th day when compared with the value on the 1st day. However, there were no significant changes for ACPACHE II score when we compared the value of day 1 and day 14 in the control and B6-100 groups. The most common diagnoses were gastrointestinal disorders (i.e., gastric ulcer, peptic ulcer, acute pancreatitis, cholecystitis, peritonitis, inflammatory bowel disease, and intestinal obstruction), malignant neoplasms (i.e., esophagus, lung, breast, stomach, duodenal, prostate, rectum and colon cancer), infection (i.e., deep neck infection and sepsis), respiratory diseases (i.e., adult respiratory distress syndrome and pneumonia), and multiple organ failure.
The mean intakes of carbohydrate, lipid, and protein for 14 days were 309.2±85.9, 63.6±35.0, and 61.2±24.4 g in the control group; 262.8±53.9, 53.5±18.3, and 47.5±15.3 g in the B6-50 group; and 290.0±50.7, 50.9±12.5, and 54.4±24.0 g in the B6-100 group. There were no significant differences in macronutrient and vitamin B6 intake (Table 3) among the three groups. Total vitamin B6 intake (dietary plus supplementation) significantly correlated with plasma PLP concentration in all subjects (r=0.56, P<0.001).
Measurements response to vitamin B6 supplementation
The results of hematological measurement are shown in Table 2. On day 1, there were no significant differences with respect to serum hemoglobin, hematocrit, albumin, prealbumin, alkaline phosphatase, creatinine, and hs-CRP among the three groups. The hematological measurements (i.e., hemoglobin, hematocrit, albumin, prealbumin, alkaline phosphatase, creatinine, and hs-CRP), on average, were either below or over normal measurements for patients in all three groups on day 1, and the values showed no significant changes by day 14. However, prealbumin in the control and B6-100 groups, alkaline phosphatase in the B6-100 group, and hs-CRP in the control and B6-50 groups either significantly increased or decreased by day 14.
Table 3 shows responses of vitamin B6 status and immune parameters to two different doses of vitamin B6 supplements for 14 days in the critically ill patients. There were no significant differences in the values of vitamin B6 status indicators (i.e., plasma PLP, EALT-AC and EALT-AC, and urinary 4-PA) among the three groups on day 1. Mean plasma PLP level showed a marginal PLP-deficient status (20–30 nmol/l) on day 1. Plasma PLP, PL, and 4-PA and urinary 4-PA significantly increased, EAST-AC and EALT-AC significantly decreased in response to 14 days of vitamin B6 supplementation in the B6-50 and B6-100 groups. It is worth noting that 100 mg/day of vitamin B6 for 14 days did not show twofold increases in plasma PLP concentration when compared with the value in the B6-50 group.
Critically ill patients had abnormal immune responses parameters (i.e., WBC, neutrophils, TLC, T and B lymphocyte and T-suppressor cell) on the first day of admission to the ICU. Critically ill patients in the B6-50 group showed a significant increase in T lymphocytes, T-helper-cell numbers and the percentage of T-suppressor cells; the B6-100 group showed significant increases in TLC, the percentage of T lymphocytes, T-helper-cell numbers, and the percentage of T-suppressor cell (Table 3). There were no significant changes in all immune parameters except for the percentage of neutrophils in the control group when compared with the values between day 1 and day 14.
Associations with immune responses
Spearman correlation coefficients were performed to understand the relation between immune responses and vitamin B6 status indicators (Table 4). Plasma PLP concentration was inversely associated with neutrophil cell numbers, but positively associated with total lymphocytes, T lymphocytes, and T-suppressor cell numbers. Plasma PL concentration was positively correlated with total lymphocytes and T-suppressor-cell numbers. Plasma 4-PA only negatively correlated with neutrophil cell numbers. The values of EALT-AC and EAST-AC were negatively correlated with total lymphocytes, T lymphocytes and T-suppressor-cell numbers.
Plasma PLP has been shown to be a significant indicator of immune responses in critically ill patients in our recent study (Huang et al., 2005). However, the effect of vitamin B6 supplementation on immune responses in immunocompromised subgroups within the population (i.e., elderly, ill patients) has been seldom reported. Only a few studies have shown that 50–300 mg/day vitamin B6 supplementation in either healthy elderly or hemodialysis patients significantly improved immune responses of subjects (Casciato et al., 1984; Talbott et al., 1987; Folkers et al., 1993). In the present study, our critically ill patients had abnormal immune responses even though their vitamin B6 intake was higher than Taiwan DRI recommendations (Department of Health, Taiwan, 2002) on the first day of admission to the ICU. In agreement with the previous studies (Casciato et al., 1984; Talbott et al., 1987; Folkers et al., 1993), several cellular immune response parameters (i.e., T lymphocyte, T-helper and T-suppressor cells) significantly increased after the supplementation of either 50 or 100 mg/day of vitamin B6 for 14 days; whereas immune responses showed no significant changes in the control group. This suggests that vitamin B6 supplementation could increase immune responsiveness of only T cells but not B cells. The increase in the percentage of T3+ and T4+ cells has been suggested to affect the differentiation of immature T cells to mature T cells by vitamin B6 supplementation in healthy elderly (Talbott et al., 1987).
In the study of Gray et al. (2004), patients who underwent an elective knee arthroplasty had a significant increase in circulatory CRP concentrations and there was a significant fall in plasma PLP concentration but red cell PLP remained stable. This may be that critically ill patients were under severe stress during the systemic inflammatory response, which may have increased the turnover and utilization of plasma PLP, decreased hepatic PLP reserves (Louw et al., 1992), or redistributed PLP from plasma to erythrocyte (Talwar et al., 2003a; Gray et al., 2004; Quasim et al., 2005). Quasim et al. (2005), therefore, suggested that direct measurements of red cell PLP are more responsive to supplementation than plasma measurements in the critically ill patients. That might be the reason why there was no correlation between vitamin B6 intake and plasma PLP concentration in our two previous studies (Huang et al., 2002; Huang et al., 2005). Critically ill patients consumed approximately 4 mg/day of vitamin B6, and a marginal plasma PLP deficiency (∼20 nmol/l) and abnormal immune responses were still observed (Huang et al., 2005). We, therefore, gave a large amount of vitamin B6 to our critically ill patients, finding total vitamin B6 intake (dietary plus supplementation) significantly correlated with plasma PLP concentration. It seemed that a large dose of vitamin B6 supplementation (50 or 100 mg/day) could compensate for the lack of responsiveness of plasma PLP to vitamin B6 intake and plasma PLP concentrations increased in the critically ill patients. The significant increased plasma PLP concentration may further increase the responses of T cells in our critically patients, although the correlation between plasma PLP and immune cells was relatively weak.
Two doses of vitamin B6 supplements (50 and 100 mg) were given daily to our critically ill patients. However, plasma PLP concentration did not correlate with dose response, which meant mean plasma PLP level of the B6-100 group did not increase two times more than plasma PLP level of the B6-50 group. In addition, parameters of immune function in the B6-100 group did not show more improvement than in the B6-50 group. We, therefore, suspected that 50 mg/day of vitamin B6 might be the saturation level for critically ill patients, who could not efficiently utilize the higher dose (i.e., 100 mg/day) of vitamin B6.
In the control group, plasma PLP concentration remained unchanged during the study period, but plasma PL significantly increased on day 14 in the ICU. A possible explanation could be that plasma PLP is bound to serum albumin while being transported by the blood; however, low serum albumin in our critically ill patients caused the dephosphorylation of plasma PLP into PL (Merrill and Henderson, 1987).
In conclusion, 50 mg/day or higher of vitamin B6 supplementation could compensate for the lack of responsiveness of plasma PLP to vitamin B6 intake, and further increase immune response of critically ill patients. Erythrocyte PLP may be more responsive to supplementation than plasma measurements in the critically ill patients (Quasim et al., 2005), and we did not measure erythrocyte PLP when assessing vitamin B6 status in this study; however, our results provide more information in the clinical practice for considering using vitamin B6 supplementation to increase the immune function of critically ill patients.
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Guarantor: YC Huang.
Contributors: C-HC was responsible for the screening and intervention of subjects and interpretation of the results. S-JC was responsible for data coding, sample analyses, and statistical analyses. B-JL was responsible for the screening and intervention of subjects. K-LL was responsible for the hematological measurements. Y-CH was responsible for the development of intellectual content and the study design, interpretation of the results, and manuscript drafting.
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Cheng, CH., Chang, SJ., Lee, BJ. et al. Vitamin B6 supplementation increases immune responses in critically ill patients. Eur J Clin Nutr 60, 1207–1213 (2006). https://doi.org/10.1038/sj.ejcn.1602439
- pyridoxal 5′-phosphate
- immune responses
- vitamin B6 supplementation
- critically ill
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